Method for automatic differential leukocyte count

ABSTRACT

A differential count of leukocytes in blood is performed automatically by passing the individual blood cells through a constricted chamber while illuminating them with sufficient ultraviolet light to cause fluorescent emissions from each cell. The respective fluorescent emissions from the different types of leukocyte cells are each characteristic of the particular type of cell and can be automatically registered using photomultiplier tubes and counters.

United States Patent Trowe METHOD FOR AUTOMATIC DIFFERENTIAL LEUKOCYTECOUNT Neil M. Trowe, Potomac, Md.

Wheeler International Inc., Washington, DC.

Filed: Aug. 24, 1970 Appl. No.: 66,560

Inventor:

Assignee:

References Cited UNITED STATES PATENTS 11/1968 Kamentsky 250/833 UV X [41 July 22,1975

3,497,690 2/1970 Wheeless, Jr. et al. 250/7] R 3,515,884 6/1970 lmadate250/218 3,549,994 12/1970 Rothermel et al. 235/92 PC PrimaryExaminer-Archie R. Borchelt Attorney, Agent, or FirmCantor & Kraft [5 7ABSTRACT A differential count of leukocytes in blood is performedautomatically by passing the individual blood cells through aconstricted chamber while illuminating them with sufficient ultravioletlight to cause fluorescent emissions from each cell. The respectivefluorescent emissions from the different types of leukocyte cells areeach characteristic of the particular type of cell and can beautomatically registered using photomultiplier tubes and counters.

3 Claims, No Drawings METHOD FOR AUTOMATIC DIFFERENTIAL LEUKOCYTE COUNTThis invention is concerned with a high speed, accurate and automatedmethod for performing a differential analysis of white blood cells. Morespecifically, the present invention is concerned with determining theindividual counts of the various types of white blood cells orleukocytes by registering their distinctive fluorescent emissions.

Hematology is an important department of the clinical pathologylaboratory. For many years pathologists have been concerned with varioushematologic procedures. Among the earliest procedures was an examinationof unstained blood; later the use of specific staining methods for theidentification of various kinds of white cells was attempted. Thenmethods of absolute blood counting were introduced together with specialtests such as, supravital staining and a method of differential countingof the various types of blood cells, introduced by Victor Schilling.

Increasingly it has become important to be able to make rapid, accurateblood tests and while at least one generally accepted method has beenderived to give hematrocrit, hemoglobin and total leukocyte counts in arapid and reasonably accurate, semi-automated manner; the method forperforming a differential count of the various kinds of leukocyte cellshas improved only in respect to the staining techniques used. No methodhas heretofore been developed which would even partially automate theleukocyte differential and bring it to the level of performance of thehematrocrit, hemoglobin or total leukocyte count.

According to present techniques for making differential leukocytecounts, a slide is prepared of a drop of blood and the specimen thenstained and dried. The various types of leukocytes (i.e. lymphocytes,eosinophils, basophils and monocytes) present different appearances whenilluminated under a microscope and accordingly a trained technician isable to make a count. Normally, a total of 100 leukocytes are counted inthis manner and the number of each type of cell counted is reported inpercentage form.

Assuming that the drop of blood placed on the slide to make the smear isof a uniform size, it is imperative that the prepared blood smear bemade in such a way that there is a feathering of the smear down into aone cell thickness. Care must be taken to assure that the smear is madewithout the blood reaching the edge of the slide. If this does occur,the large cells are concentrated at the edge of the smear and thesmaller cells are concentrated in the middle of the smear. An accuratedifferential on a smear of this type is impossible. In the stainingtechnique itself, there are also many points at which error can occur.One of the most common errors at this point is in using a buffer for thestaining which is either too alkaline or too acid; the buffer and stainmixture must be neutral. If the buffer is either too acid or tooalkaline, the possibility of determining the maturity of the leukocyteis greatly reduced because of the non-uniform staining of the nucleus.

Now in accordance with the present invention it has been found that boththe total and differential count of leukocytes in blood can be quickly,efficiently, and accurately determined automatically without the needfor technicians or other personnel visually observing the cells orotherwise manually making the determination and consequently with agreat reduction in the possibility of error. i

More specifically, it has been found that the various types of whiteblood cells or leukocytes i.e. lymphocytes, monocytes, and granulocytesincluding neutrophils, basophils, and eosinophils each exhibitcharacteristic fluorescence and can be excited with ultravioletradiation so each emits fluorescent radiation having a wave lengthcharacteristic of that particular type of cell. Further it has beenfound that using a spectrofluorometor, the respective fluorescentemissions from the different types of white blood cells can be recordedin such a way as to permit the mechanical counting of the number of eachtype of cell in a given volume.

According to the present invention, a blood sample which has beensuitably diluted with about 40 50 parts by volume of say about a 1percent saline solution is treated, for example with an appropriatelysing agent to destroy the erythrocytes present. Subsequently thetreated specimen is introduced into a chamber or tube which narrows to asufficiently small diameter, e.g. about microns, so that the passage ofcells is in single file by one or more beams of ultravioletmonochromatic light of sufficient intensity to excite the cells tofluorescent emission. These fluorescent emissions from the respectiveilluminated cells are segregated according to the wavelengthscharacteristic of each cell and counted on photomultiplier tubes.Segregation of the fluorescent emissions from the respective cellsaccording to wavelength can be effected by means of an analyzermonochrometor having photomultiplier tubes located at slits positionedto discriminate the wavelengths of the emissions of each of thedifferent types of white blood cells being counted. Alternatively, forexample, dicroic minors can also be arranged to receive and separate therespective wavelengths emitted from the cells.

The ultraviolet illumination from the exciter monochrometor causes thewhite blood cells to fluoresce and the fluorescence is detected by thephotomultiplier tubes at the analyzer exit slits. The output pulse fromthe photomultiplier tubes is passed to counters corresponding to thespecific white cell being detected. In addition, the outputs of each ofthe photomultipliers is passed to a total white cell counter. When thetotal white cell counter indicates a total of counts, all counters canbe stopped, their counts now indicating the percentage abundance of eachof the cell types.

EXAMPLE 0.1 c.c. of whole blood diluted in 4.9 c.cs of 0.9 percentaqueous sodium cloride solution was added to one drop of lysing agent todestroy the erythrocytes. After mixing thoroughly, the solution waspoured into the mouth of a vessel provided with a release valve and avacuum applied which caused the blood to pass into the testing chamberwhich narrowed to a 70 micron diameter orifice and then into a 70 microntube which was placed at the exit slit of an exciter monochrometor. Thecells flowed through in single file and were illuminated by amonochromatic beam of light. At right angles to the path of the exciterbeam was the entrance slit to an analyzer monochrometor. At theappropriate wavelength positions for each of the different types ofwhite blood cells to be counted, photomultiplier tubes were located.

The ultraviolet illumination from the exciter monochrometor caused thewhite blood cells to fluoresce and the characteristic fluorescence ofthe individual e. registering the respective fluorescent emissions cellswas detected by the individual photomultiplier from each leukocyte cellaccording to its wavetubes at the analyzer exit slits. The output pulsefrom length on a photomultiplier tube; the photomultiplier tubes waspassed to counters corref. by means of counters recording the number offluosponding to the specific white cells being detected. In 5 rescentemissions registered on the photomultiplier addition, the outputs ofeach of the photomultipliers tubes from each type of leukocyte cell. waspassed to a total white cell counter. When the total 2. The method ofclaim 1, wherein the leukocyte cells white cell counter indicated atotal of 100 counts, all are lymphocytes, monocytes and granulocytes.counters were stopped so that their counts indicated 3. A method formaking total and differential counts the actual percentage abundance ofeach of the cell of the leukocytes in blood which comprises the stepsof: types. a. destroying the erythrocytes in a sample of the What isclaimed is: blood cells; 1. A method for making a differential count ofleukob. subjecting said sample to ultra-violet radiation sufcytes inblood which comprises the steps of: ficient to cause fluorescentemissions of the leuko' a. preparing a dilute saline solution of theblood samcytes therein;

ple; c. separating the resulting emissions according to b. passing thedilute saline blood solution into a conwavelengths into lymphocytes,monocytes and stricted chamber so that the individual cells aredisgranulocytes emissions sets; posed in a single array; d. registeringthe number of emissions for each set 0. subjecting the cells disposed insaid constricted and for the sum of said sets; and

chamber in single file to sufficient ultraviolet radiae. stoppingregistration of said emissions when the tion as the cells pass throughthe chamber to cause total leukocyte count reaches 100; whereby saidthem to fluoresce; number of emissions for each set indicates the perd.segregating the fluorescent emissions from each centage abundance ofsaid set relative to said total cell according to the characteristicwavelength of leukocyte count.

the fluorescent emission of that type of cell;

Disclaimer 3,896,307.Neil M Trowe, Potomac, Md. METHOD FOR AUTOMATICDIF- FERENTIAL LEUKOCYTE COUNT. Patent dated July 22, 1975. Disclaimerfiled Feb. 13, 1981, by the assignee, Wheeler International,

Inc. Hereby enters this disclaimer to claims 1-3, all the claims of saidpatent.

[Official Gazette April 14, 1981.]

1. A method for making a differential count of leukocytes in blood whichcomprises the steps of: a. preparing a dilute saline solution of theblood sample; b. passing the dilute saline blood solution into aconstricted chamber so that the individual cells are disposed in asingle array; c. subjecting the cells disposed in said constrictedchamber in single file to sufficient ultraviolet radiation as the cellspass through the chamber to cause them to fluoresce; d. segregating thefluorescent emissions from each cell according to the characteristicwavelength of the fluorescent emission of that type of cell; e.registering the respective fluorescent emissions from each leukocytecell according to its wavelength on a photomultiplier tube; f. by meansof counters recording the number of fluorescent emissions registered onthe photomultiplier tubes from each type of leukocyte cell.
 2. Themethod of claim 1, wherein the leukocyte cells are lymphocytes,monocytes and granulocytes.
 3. A method for making total anddifferential counts of the leukocytes in blood which comprises the stepsof: a. destroying the erythrocytes in a sample of the blood cells; b.subjecting said sample to ultra-violet radiation sufficient to causefluorescent emissions of the leukocytes therein; c. separating theresulting emissions according to wavelengths into lymphocytes, monocytesand granulocytes emissions sets; d. registering the number of emissionsfor each set and for the sum of said sets; and e. stopping registrationof said emissions when the total leukocyte count reaches 100; wherebysaid number of emissions for each set indicates the percentage abundanceof said set relative to said total leukocyte count.